Abstract:

The present invention provides fluorescent nucleoside analogs with
conjugated membered heterocycles, including furan and thiophene. The
fluorescent nucleoside analogs maintain structural similarity to
naturally occurring nucleoside bases, mimicking shape, size,
hybridization, and recognition properties. Incorporation of the
fluorescent cyclic compounds confers specific photophysical
characteristics including a bathochromic (red) shift of the absorption
spectrum to minimize absorption overlap with naturally occurring
nucleoside bases, and a shift to the long emission wavelength in the
visible range. The invention also provides for various methods of
synthesizing the fluorescent nucleoside analogs and incorporating the
fluorescent analogs in DNA, RNA, or oligomer synthesis. Further, methods
of detecting the fluorescent nucleoside analogs in an oligonucleotide or
oligomer are provided. The subject compounds are useful as probes in the
study of the structure and dynamics of nucleic acids and their complexes
with proteins.

Claims:

1. A compound having the general formula (I):wherein dashed lines
represent optional double bonds;A and B are, independently, --CH═,
--O--, or --S--, and A and B are different where ever they appear;C and D
are, independently, --N═ or --CH═; andZ is --NH2 or
═O,with the proviso that when A is --O-- or --S--, B is not --O-- or
--S--, and when C is --N═, D is not --N═;R is --H or a glycal
having the general formula (II)a or (II)b:R1 is --H, --PO3,
orand R2 is --H, --PO3, oror salts thereof.

2. The compound of claim 1, wherein A is --O--, B, C, and D are --C═,
Z is ═O, and R is

3. The compound of claim 1, wherein A is --S--, B, C, and D are --C═,
Z is ═O, and R is

4. The compound of claim 1, wherein A is --O--, D is --N═, Z is
═O, and R is

5. The compound of claim 1, wherein A is --S--, D is --N═, Z is
═O, and R is

15. A compound having the general formula (III):wherein each of X and Y
is, independently, --O--, --S--, or --CH═, and X and Y are different
where ever they appear, with the proviso that when X is --O-- or --S--, Y
is not --O-- or --S--;Z is selected from --CH═, --N═, or
--CR.sup.1.dbd.; andring B is selected from:wherein R1 and R2
are each the same or different, where ever they appear, and each is
selected from --H or a glycal having the general formula (II)a or
(II)b:R3 is --H, --PO3, or:and R4 is --H, --PO3,
or:with the proviso that when R1 is a glycal, R2 is not a
glycal;or salts thereof.

28. A kit comprising:at least one compound of claim 1 or an
oligonucleotide comprising the at least one compound;a container;
anddirections for using the at least one compound or oligonucleotide.

29. The kit of claim 28, wherein the at least one compound is a
phosphoramidite derivative.

30. A kit comprising:at least one compound of claim 15 or an
oligonucleotide comprising the at least one compound;a container;
anddirections for using the at least one compound or oligonucleotide.

31. The kit of claim 30, wherein the at least one compound is a
phosphoramidite derivative.

32. A method of synthesizing pyrimidine analogs comprisinga) admixing
5-iodo-2'-deoxyuridine or 3',5'-diTol-Iodo-dU and the corresponding
stannylated heterocycles in the presence of palladium;b) protecting the
5'-hydroxyl with 4,4'-dimethoxytrityl chloride; andc) phosphitylating the
unprotected 3'-hydroxyl.

33. A method of synthetically preparing a fluorescently labeled
oligonucleotide comprising incorporating at least one compound of claim 1
or 15 into a DNA or RNA chain.

34. The method of claim 33, further comprising admixing the at least one
compound with a growing DNA or RNA chain, wherein the at least one
compound is a phosphoramidite derivative.

35. The method of claim 34, further comprising synthesis on a solid phase.

36. A method of detecting a target nucleic acid in a sample
comprising:contacting the sample with one or more oligonucleotides having
at least one compound of claim 1 or 15 incorporated therein, for a time
and under conditions sufficient to allow hybridization to occur between
the target nucleic acid and the oligonucleotides;separating
non-hybridized oligonucleotides;exciting the hybridized oligonucleotides;
anddetecting fluorescence produced by complexes formed between the
oligonucleotides and the target nucleic acid,wherein detecting
fluorescence correlates with the presence of the target nucleic acid.

38. The method of claim 36, wherein the one or more oligonucleotides are
immobilized on a solid phase.

39. The method of claim 38, wherein the one or more oligonucleotides are
positioned on an array.

40. A compound having the general formula (IV):where, X and Y are,
independently, --CH═, or --O--, and X and Y are different where ever
they appear, with the proviso that when A is --O--, B is not --O--,R is
--H or a glycal having the general formula (II)a or (II)b:R1 is --H,
--PO3, orand R2 is --H, --PO3, oror salts thereof.

41. A compound having the general formula (V):wherein R is --H or a glycal
having the general formula (II)a or (II)b:R1 is --H, --PO3,
orR2 is --H, --PO3, orR3 and R4 are each
independently --H or a furan having the general formula (VI):wherein each
R3 and R4 is different where ever they appear with the proviso
that when R3 is Formula (VI), R4 is not Formula (VI);or salts
thereof.

[0003]This invention relates to fluorescent nucleoside analogs as probes
for nucleic acid structure, dynamics, and function as well as sequence
and lesion analysis.

BACKGROUND INFORMATION

[0004]Fluorescence methods are extremely widespread in chemistry and
biology. The methods give useful information on sequence, structure,
distance, orientation, complexation, location for biomolecules, and
measurements of dynamics and kinetics. As a result, many strategies for
fluorescence labeling of biomolecules, including nucleic acids, have been
developed.

[0005]For example, in the case of DNA, a convenient and useful method for
fluorescence labeling is to add a fluorescent moiety during the DNA
synthesis itself. However, the majority of labels commonly used during
DNA synthesis are attached to the DNA as tethers that are often 5 to 11
or more atoms long. These tethers can be problematic, for example, the
tethers make the location of the dye difficult to determine precisely,
interfere with DNA-protein interactions, etc.

[0006]Unfortunately, fluorescent nucleosides that mimic naturally
occurring nucleoside bases structurally and chemically are scarce. Thus,
a need exists for fluorescent nucleosides which are structurally and
chemically similar to naturally occurring nucleosides. The present
invention provides nucleoside analogs with improved photophysical
characteristics and the subject nucleoside analogs are more generally
useful in biophysical and diagnostics applications.

[0008]The present invention also relates to fluorescent analogs containing
conjugated 5-membered heterocycles which maintain the structural
similarity to that of naturally occurring nucleoside bases (i.e., purines
and pyrimidines), including substantially similar shape, sizes,
hybridization and recognition capabilities. Further the fluorescent
analogs of the invention have advantageous photophysical characteristics
over that of the naturally occurring nucleoside bases, including emission
spectrum in the longer wavelengths (i.e., towards the visible range),
bathochromatic (red) shifted absorption spectrum such that there is
minimization of overlap with the naturally occurring nucleoside bases.

[0009]In one embodiment, a compound is provided having the general formula
(I):

where dashed lines represent optional double bonds, A and B are,
independently, --CH═, --O--, or --S--, and A and B are different
where ever they appear, C and D are, independently, --N═ or
--CH═, and Z is --NH2 or ═O, with the proviso that when A is
--O-- or --S--, B is not --O-- or --S--, and when C is --N═, D is not
--N═, R is --H or a glycal having the general formula (II) a or (II)b

R1 is --H, --PO3, or

[0010]and R2 is --H, --PO3, or

[0011]or salts thereof.

[0012]In one aspect, A is --O--, B, C, and D are --C═, Z is ═O,
and R is

[0013]In another aspect, A is --S--, B, C, and D are --C═, Z is
═O, and R is

[0014]In a further aspect, A is --O--, D is --N═, Z is ═O, and R
is

[0015]In another aspect, A is --S--, D is --N═, Z is ═O, and R is

[0016]In another embodiment, a synthetic oligonucleotide is provided
including at least one compound of having the general formula (I), where
the synthetic oligonucleotide substantially hybridizes to a complementary
naturally occurring polynucleotide or oligonucleotide, including where
the synthetic oligonucleotide, naturally occurring polynucleotide, and
naturally occurring oligonucleotide comprises DNA or RNA.

[0017]In one embodiment, a compound is provided having the general formula
(III):

where each of X and Y is, independently, --O--, --S--, or --CH═, and X
and Y are different where ever they appear, with the proviso that when X
is --O-- or --S--, Y is not --O-- or --S--, Z is selected from --CH═,
--N═, or --CR1═, and ring B is selected from:

where R1 and R2 are each the same or different, where ever they
appear, and each is selected from --H or a glycal having the general
formula (II)a or (II)b:

R3 is --H, --PO3, or:

[0018]and R4 is --H, --PO3, or:

[0019]with the proviso that when R1 is a glycal, R2 is not a
glycal;or salts thereof.

[0020]In one aspect, X is --O--, Z is --CR1═, ring B is

[0021]and R1 is

[0022]In another aspect, X is --S--, Z is --CR1═, ring B is

and R1 is

[0024]In a further aspect, X is --S--, Z is --CH═, ring B is

and R2 is

[0026]In another embodiment, a synthetic oligonucleotide is provided
including at least one compound having the general formula (III), where
the synthetic oligonucleotide substantially hybridizes to a complementary
naturally occurring polynucleotide or oligonucleotide, including where
the synthetic oligonucleotide, naturally occurring polynucleotide, and
naturally occurring oligonucleotide comprises DNA or RNA.

[0027]In one embodiment, a compound is disclosed having the general
formula (IV):

where, X and Y are, independently, --CH═, or --O--, and X and Y are
different where ever they appear, with the proviso that when X is --O--,
Y is not --O--, R is --H or a glycal having the general formula (II)a or
(II)b:

R1 is --H, --PO3, or

[0028]and R2 is --H, --PO3, or

[0029]or salts thereof.

[0030]In one embodiment, a compound is disclosed having the general
formula (V):

where R is --H or a glycal having the general formula (II)a or (II)b:

R1 is --H, --PO3, or

[0031]R2 is --H, --PO3, or

[0032]R3 and R4 are each independently --H or a furan having the
general formula (VI):

[0033]where each is different where ever they appear with the proviso
that [0034]when R3 is Formula (VI), R4 is not Formula (VI);or
salts thereof.

[0035]In one embodiment, kits are described, including at least one
compound of the general formula (I) or (III) or an oligonucleotide
comprising the at least one compound of the general formula (I) or (II),
a container, and directions for using the at least one compound or
oligonucleotide. In a related aspect, the at least one compound is a
phosphoramidite derivative.

[0036]In another embodiment, a method of synthesizing 5-modified
pyrimidine analogs is provided comprising admixing 5-iodo-2'-deoxyuridine
or 3',5'-diTol-Iodo-dU and the corresponding stannylated heterocycles in
the presence of palladium, protecting the 5'-hydroxyl with
4,4'-dimethoxytrityl chloride, and phosphitylating the unprotected
3'-hydroxyl.

[0037]In one embodiment, a method is disclosed for synthetically preparing
a fluorescently labeled oligonucleotide including incorporating at least
one compound of the general formula (I) or (III) into a DNA or RNA chain.
In a related aspect, the method further comprises admixing the at least
one compound with a growing DNA or RNA chain, where the at least one
compound is a phosphoramidite derivative, including synthesis on a solid
phase.

[0038]In another embodiment, a method is disclosed for detecting a target
nucleic acid in a sample including, contacting the sample with one or
more oligonucleotides having at least one compound of the general formula
(I) or (III) incorporated therein, for a time and under conditions
sufficient to allow hybridization to occur between the target nucleic
acid and the oligonucleotides, separating non-hybridized
oligonucleotides, exciting the hybridized oligonucleotides, and detecting
fluorescence produced by complexes formed between the oligonucleotides
and the target nucleic acid, where detecting fluorescence correlates with
the presence of the target nucleic acid.

[0039]The invention also provides methods for synthesizing the fluorescent
nucleoside analogs that maintain structural similarity to naturally
occurring nucleoside bases and with conjugated fluorescent 5-membered
heterocyles. These methods include cross-coupling the heterocyle to the
naturally occurring nucleoside, N-glycosylation the heterocyle to the
naturally occurring nucleoside, and C-glycosylation the heterocyle to the
naturally occurring nucleoside. Other methods of synthesizing the
fluorescent analogs which is known or standard in the art, or which will
become known or standard in the art is anticipated and within the scope
of the present invention.

[0040]The invention also provides methods of preparing fluorescently
labeled nucleic acid molecules incorporating at least one fluorescent
nucleoside analog of the present invention, for example into an RNA or
DNA molecule under conditions sufficient to incorporate the fluorescent
nucleoside analog. Similarly, the invention provides for nucleotide
analogs comprising one or more fluorescent nucleoside analogs of the
present invention.

[0041]The invention also provides methods of detecting a target nucleic
acid molecule in a sample to be tested by contacting the target nucleic
acid with a nucleic acid probe containing at least fluorescent nucleoside
analog for time and under conditions sufficient to permit hybridization
between the target nucleic acid molecule and the fluorescent probe and
detecting the hybridization.

[0042]The invention also provides for an array containing multiple solid
supports and multiple locations on a solid support where each support or
location has attached an oligomer containing the fluorescent nucleoside
analogs.

[0045]FIG. 3 shows selected synthetic routes utilized for the synthesis of
modified nucleosides. FIG. 3A: Synthesis of a modified N-nucleoside 7;
and FIG. 3B: Synthesis of a C-nucleoside 6 where the brominated
heterocycle is cross-coupled to a glycal.

[0046]FIG. 4 is a graph showing the emission spectra of nucleoside 1 in
various solvents ranging from water (most polar) to diethyl ether (least
polar).

[0047]FIG. 5 is a graph illustrating the hyperchromism (enhanced emission)
and bathochromic (red) shift displayed by nucleoside 1 and its
sensitivity ("responsiveness") to environmental changes.

DETAILED DESCRIPTION OF THE INVENTION

[0048]Fluorescent nucleoside analogs with high emission quantum efficiency
and long emission wavelength are usually associated with significant
structural and chemical modifications when compared to their natural
counterparts. The major challenge in this field is, therefore, to design
nucleoside analogs with "optimal" photophysical characteristics (e.g.,
red-shifted absorption and emission spectra and highest possible emission
quantum yield) while maintaining high structural homology to the
naturally occurring nucleoside bases.

[0049]Nucleotides or oligonucleotides or oligomers of the present
invention, comprising naturally occurring nucleotides and phosphodiester
bonds can be chemically synthesized or can be produced using recombinant
DNA methods, using an appropriate polynucleotide as a template. In
comparison, an oligonucleotide comprising nucleotide analogs or covalent
bonds other than phosphodiester bonds generally will be chemically
synthesized, although an enzyme such as T7 polymerase can incorporate
certain types of nucleotide analogs into an oligonucleotide and,
therefore, can be used to produce such an oligonucleotide recombinant
from an appropriate template (Jellinek et al., supra, 1995).

[0051]In one embodiment, a compound is disclosed having the general
formula (I):

where dashed lines represent optional double bonds, A and B are,
independently, --CH═, --O--, or --S--, and A and B are different
where ever they appear, C and D are, independently, --N-- or --CH═,
and Z is --NH2 or ═O, with the proviso that when A is --O-- or
--S--, B is not --O-- or --S--, and when C is --N═, D is not
--N═, R is --H or a glycal having the general formula (II)a or (II)b:

R1 is --H, --PO3, or

[0052]and R2 is --H, --PO3, or

[0053]or salts thereof.

[0054]In one aspect, A is --O--, B, C, and D are --C═, Z is ═O,
and R is

[0055]In another aspect, A is --S--, B, C, and D are --C═, Z is
═O, and R is

[0059]In another embodiment, a compound is provided having the general
formula (III):

where each of X and Y is, independently, --O--, --S--, or --CH═, and X
and Y are different where ever they appear, with the proviso that when X
is --O-- or --S--, Y is not O-- or --S--, Z is selected from --CH═,
--N═, or --CR1═, and ring B is selected from:

where R1 and R2 are each the same or different, where ever they
appear, and each is selected from --H or a glycal having the general
formula (II)a or (II)b:

R3 is --H, --PO3, or:

[0060]and R4 is --H, --PO3, or:

[0061]with the proviso that when R1 is a glycal, R2 is not a
glycal; or salts thereof.

[0069]In one embodiment, a compound is disclosed having the general
formula (IV):

where, X and Y are, independently, --CH═, or --O--, and X and Y are
different where ever they appear, with the proviso that when X is --O--,
Y is not --O--,R is --H or a glycal having the general formula (II)a or
(II)b:

R1 is --H, --PO3, or

[0070]and R2 is --H, --PO3, or

[0071]or salts thereof.

[0072]In one embodiment, a compound is disclosed having the general
formula (V):

where R is --H or a glycal having the general formula (II)a or (II)b:

R1 is --H, --PO3, or

[0073]R1 is --H, --PO3, or

[0074]R3 and R4 are each independently --H or a furan having the
general formula (VI):

[0075]where each is different where ever they appear with the proviso
that [0076]when R3 is Formula (VI), R4 is not Formula (VI);or
salts thereof.

[0077]Further, compounds of the invention include:

[0078]Nucleotide 8 is the dC analog of the modified T(dU) that has been
synthesized by the methods disclosed herein. It is emissive
(λem 443 nm φ˜1%). Nucleoside 9 is analog of 8,
where the furan is fused to a new pyrrole ring (while maintaining the
H-bonding capability of C). Nucleoside 10 is an isomer of 9, where the
connectivity is different. Nucleosides 11 and 12 represent fused analogs
of C, where a furan is conjugated but not fused to the pyrrole ring.

[0079]Further, compounds provided in the present disclosure possess a
red-shifted absorption spectrum which does not substantially overlap with
the absorption spectrum of a naturally occurring nucleoside, where the
absorption spectrum is in the range of about 240 nm to about 350 nm,
about 250 to about 320, about 262 to about 318, about 266 to about 294,
about 268 to about 293, or about 286 to about 298.

[0080]In a related aspect, compounds provided in the present disclosure
possess an emission spectrum in the range of about 300 to about 450,
about 335 to about 435, about 337 to about 433, about 339 to about 431,
or about 412 to about 413.

[0081]In one aspect, such compounds posses a long emission wavelength in
the visible spectrum.

[0082]Fluorescent nucleoside analogs of the present invention are
sensitive to their local environment. They can be studied using real
time, sensitive assays for nucleic acids structure, dynamics and
recognition. Assays measuring and detecting the fluorescent nucleoside
analogs of the invention have many applications because they simplify and
accelerate the accumulation of data pertinent to a specific recognition
phenomenon (e.g., DNA-protein interaction, RNA-small molecule
interaction). For example, in the pharmaceutical industry, such assays
are essential for high throughput screening protocols, particularly in
the context of drug discovery. Other applications, include studying
nucleic acid modifying enzymes (e.g., DNA methyl transferases,
polymerases, helicases, RNA modifying enzymes such as dicer, etc.) that
play crucial roles in development, genetic diseases and cancers, the
discovery of novel anti-HIV agents assisted by fluorescent TAR
constructs, and the discovery of novel antibiotics targeted at the
bacterial ribosome assisted by a fluorescent A-site analog, etc.

[0083]In one embodiment, a synthetic oligonucleotide is provided,
including at least one compound of general formula (I) or general formula
(III), where the synthetic oligonucleotide substantially hybridizes to a
complementary naturally occurring polynucleotide or oligonucleotide. In a
related aspect, the synthetic oligonucleotide, naturally occurring
polynucleotide, and naturally occurring oligonucleotide comprise DNA or
RNA.

[0084]In another embodiment, a kit is disclosed including at least one
compound of the general formula (I) or general formula (II) or an
oligonucleotide comprising the at least one compound, a container, and
directions for using the at least one compound or oligonucleotide. In a
related aspect, the at least one compound is a phosphoramidite
derivative.

[0085]In one embodiment, synthetic routes are provided, according to
Schemes 1-4:

[0086]Synthesis of Heterocycle:

[0087]Synthesis of Ribonucleoside:

[0088]Synthesis of 2'-deoxyribonucleoside:

[0089]Synthesis of 2'-deoxyribonucleoside Phosphoramidite:

[0090]In another embodiment, synthetic routes are provided according to
Schemes 5 and 6:

[0091]Note the use of building blocks where the fully modified dU
derivative can be effectively converted into the dC analog.

[0094]In one embodiment, a method is disclosed for synthetically preparing
a fluorescently labeled oligonucleotide comprising incorporating at least
one compound of the general formula (I) or general formula (III) into a
DNA or RNA chain. In a related aspect, the at least one compound is
admixed with a growing DNA or RNA chain, where the at least one compound
is a phosphoramidite derivative. In another related aspect, such
synthesis further comprises synthesis on a solid phase.

[0095]The present compositions allow for the detection of a target nucleic
acid molecule, when present, in a sample. The target nucleic acid
molecule can be any nucleic acid molecule that can selectively hybridize
to a toehold domain of a damping oligonucleotide, particularly a damping
oligonucleotide of a component of a translator. The target sequence can
be a gene sequence or portion thereof (e.g., a transcriptional and/or
translational regulatory sequence, coding sequence, or intron-exon
junction), a cDNA molecule, an RNA (e.g., an mRNA, tRNA or rRNA), or any
other nucleic acid molecule, which can be an isolated nucleic acid
molecule or a nucleic acid molecule contained in a sample (e.g., a cell
sample, wherein the target nucleic acid molecule is an endogenously
expressed molecule or is an exogenously introduced nucleic acid molecule
or expressed from an exogenously introduced molecule), and can be a
naturally occurring nucleic acid molecule or a synthetic molecule. A
target sequence can be any length, provided that selective hybridization
with a toehold domain can occur. A target sequence also can be contained
within a larger nucleic acid molecule (e.g., a restriction fragment of
genomic DNA).

[0096]The sample can be any sample that can contain a nucleic acid
molecule, including, for example, a biological sample, environmental
sample, or chemical sample. For example, a biological sample can be a
cell, tissue, or organ sample, e.g., a cell sample of an established cell
line, or a tissue sample obtained from a subject (e.g., via a biopsy
procedure), or a biological fluid sample, and can be a sample of
eukaryotic or prokaryotic origin, including a eukaryotic cell sample that
is being examined, for example, for a target sequence of an infecting
microorganism. An environmental sample that can be examined for the
presence (or amount) of a target nucleic acid molecule can be, for
example, a forensic sample (e.g., a blood sample or hair sample from a
crime scene), a water or soil sample (e.g., to identify the presence of a
contaminating organism), or a washing of a solid surface (e.g., a
hospital surface to be examined for the presence of an infectious
organism such as an antibiotic resistant bacterium).

[0097]The compositions and methods of the invention utilize selective
hybridization between a target nucleic acid molecule and a probe
containing a fluorescent nucleoside analog of the present invention.
Selective hybridization includes the specific interaction of a sequence
of a first polynucleotide with a complementary sequence of a second
polynucleotide (or a different region of the first polynucleotide). As
disclosed herein, selective hybridization of a damping oligonucleotide
and a propagating oligonucleotide can generate amplifier nucleic acid
molecules and translators, including complexes of two oligonucleotides,
three oligonucleotides, four oligonucleotides, or more. As used here, the
term "selective hybridization" or "selectively hybridize" refers to an
interaction of two nucleic acid molecules that occurs and is stable under
moderately to highly stringent conditions. The conditions required to
achieve a particular level of stringency are well known and routine, and
will vary depending on the nature of the nucleic acids being hybridized,
including, for example, the length, degree of complementarity, nucleotide
sequence composition (for example, relative GC:AT content), and nucleic
acid type, i.e., whether the oligonucleotide or the target nucleic acid
sequence is DNA or RNA.

[0098]As used herein, such "conditions and time sufficient to allow for
hybridization to occur" include, but are not limited to, conditions for
hybridization and washing under which nucleotide sequences at least
60-70% homologous to each other typically remain hybridized to each
other. The conditions can be such that sequences at least about 60%, at
least about 70%, or at least about 80%, or more homologous to each other
typically remain hybridized to each other. Such conditions are known to
those skilled in the art and can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One
example of hybridization conditions are hybridization in 6× sodium
chloride/sodium citrate (SSC) at about 45° C., followed by one or
more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Examples of
moderate to low stringency hybridization conditions are well known in the
art.

[0099]In one embodiment, a method of detecting a target nucleic acid in a
sample is provided including, but not limited to, contacting the sample
with one or more oligonucleotides having at least one compound of the
general formula (I) or the general formula (II) incorporated therein, for
a time and under conditions sufficient to allow hybridization to occur
between the target nucleic acid and the oligonucleotides, separating
non-hybridized oligonucleotides, exciting the hybridized
oligonucleotides, and detecting fluorescence produced by complexes formed
between the oligonucleotides and the target nucleic acid, where detecting
fluorescence correlates with the presence of the target nucleic acid.

[0100]In a related aspect, the target nucleic acid comprises RNA or DNA.
Further, the one or more oligonucleotides may be immobilized on a solid
phase or may be free in solution. Moreover, such one or more
oligonucleotides may be positioned on an array.

[0101]All documents provided herein are incorporated by reference in their
entirety.

[0102]The following examples are intended to illustrate but not limit the
invention.

EXAMPLE 1

[0103]The invention methods conjugate 5-membered heterocycles to
pyrimidines and purines. FIG. 1 shows fluorescent nucleoside chemical
structures which are possible. The fluorescent nucleosides can be divided
into subgroups. For example, Group I (A-F) are furo- and thieno-purine
fluorescent analogs, Group II (G-L) are 5-modified pyrimidine fluorescent
analogs, and Group III (M-P) are furo-, thieno-, and oxazolo-pyrimidine
fluorescent analogs. The listing of purine and pyrimidine fluorescent
analogs herein is not exhaustive and other conjugated 5-membered
heterocyles synthesized is well within the scope of the present
invention.

[0104]FIG. 2 shows fluorescent nucleoside structures 1-7 which have been
synthesized and evaluated for their photophysical properties, including
their absorption and emission spectrum. Fluorescent nucleoside structures
1-7 fluoresce and show a shift towards the red wavelength spectrum and
their emission spectrum is also in a long emission wavelength. For
example, fluorescent nucleoside structures 1-7 emission spectrum is in
the visible wavelength range.

EXAMPLE 2

[0105]There are several methods for synthesizing fluorescent nucleoside
structures of the invention. For example, fluorescent nucleoside
structures 14 are prepared from halogenated pyrimidines via
cross-coupling reactions. FIG. 3 shows at least two examples of
fluorescent nucleoside synthesis. In one synthetic reaction, a modified
N-nucleoside pyrimidine analog (e.g., structure 7) is synthesized using
standard N-glycosylation. In another method, C-glycosides (e.g.,
structure 6) are synthesized by a cross coupling reaction using glycals
and brominated heterocycles. The synthesis of structure 6 and 7 require
starting material structure 8. Structure 8 is synthesized by standard
transformation from simple heterocyclic precursors as shown in FIGS. 1
and 2.

[0107]Individual absorption and emission wavelengths of fluorescent
nucleoside analogs 1-7 are shown in Table I. For fluorescent nucleoside
analogs 1-7, the absorption spectra range is from about 250 to about 320
nm; and the emission spectra range is from about 337 to about 433 nm. The
absorption and emission spectra varies depending on various properties,
including the chemical structures, for example, the R group.

[0108]For some fluorescent nucleoside analogs, time resolved experiments
are performed to determine the excited state lifetime of the chromophore.
For example, fluorescent nucleoside structure 1, for example, displays a
1 nano second excited state lifetime. This excited state lifetime is
expected for an organic chromophore of this type. Further, quantum yield
measurements demonstrate a range from about 1% to above 5%.

[0109]FIG. 4 shows an emission spectra for fluorescent nucleoside analog 1
in various solvents, including water (most polar), methanol,
acetonitrile, dichloromethane, and diethyl ether (least polar).
Fluorescent nucleoside analog 1 exhibits a hyperchromism (enhanced
emission) and bathochromic (red) shift upon increasing solvent polarity.
The enhanced emission and bathochromic shift is advantageous because this
method distinguishes between a buried heterocycle (such as base paired
and base stacked nucleobase within a perfect DNA/RNA duplex) and a
solvent exposed heterocycle (upon, for example, bulging out of a
nucleobase). The enhanced emission and bathochromic shift also
distinguishes the fluorescent nucleoside analogs from that emission and
no-shift phenomenon of naturally occurring nucleoside bases.

EXAMPLE 4

[0110]Two fluorescent nucleoside analogs, 6 and 7, are converted into
their phosphoramidites derivative and incorporated into DNA
oligonucleotides using standard solid-phase DNA synthesis. The purified
oligonucleotides are characterized to ensure the incorporation of the
intact modified fluorescent nucleoside base. Hybridization reactions
followed by thermal denaturation experiments are performed to determine
structural and chemical integrity of the modified fluorescent nucleoside
base. It is shown that the incorporation of the modified fluorescent
nucleoside bases in the DNA oligonucleotides, form stable duplexes.
Therefore, the fluorescent nucleoside analogs of the present invention
are capable of hybridizing to complementary oligonucleotides similar to
naturally occurring nucleoside bases.

EXAMPLE 5

[0111]To determine the structural and chemical integrity of the
fluorescent nucleoside bases, other photophysical characteristics are
examined by steady state absorption and emission spectroscopy. FIG. 5,
shows the sensitivity of the fluorescent nucleoside bases to their
environment (e.g., absorption, emission, denaturation, etc.). These
tested parameters are routinely conducted using other techniques. One
advantage of this invention is that the thermal denaturation of the
oligonucleotides (with incorporated fluorescent nucleoside bases) is
determined by monitoring the emission spectra. FIG. 5 shows a standard
thermal denaturation curve of a duplex (5'-GCG ATG 1 ATG GCG-3') (SEQ ID
NO: 1). (5'-CGC TAC A CAT CGC-3') (SEQ ID NO: 2) containing fluorescent
nucleoside structure 1 as followed by absorbance at 260 nm next to a
curve determined by following the changes in fluorescence intensity of
fluorescent nucleoside structure 1. As shown in FIG. 5, both curves yield
approximately the same melting temperature (Tm=56° C.). The
above result was measured in 10 mM phosphate buffer, pH 7, 100 mM NaCl, 1
HM duplex.

EXAMPLE 6

[0112]Described below are the synthesis and photophysical characteristics
of a series of simple and responsive thymidine analogs where a 2'-deoxy-U
core is conjugated to aromatic 5-membered heterocycles, including furan,
thiophene, oxazole and thiazole. Synthesis of nucleosides 2a-d and
amidite 4 is shown in Scheme 7.

[0114]The one-step synthesis of the modified pyrimidines is
straightforward (Scheme 7). It entails a palladium-mediated cross
coupling of the commercially available 5-iodo-2'-deoxyuridine (or
3',5'-diTol-Iodo-dU) and the corresponding stannylated heterocycles.
Standard protection of the 5'-hydroxyl with 4,4'-dimethoxytrityl chloride
followed by phosphitylation of the unprotected 3'-hydroxyl affords the
building blocks necessary for solid-phase DNA synthesis (Scheme 7).

[0116]To evaluate the nucleoside's potential to respond to polarity
changes, their photophysical characteristics have been evaluated in
different solvents. Increasing solvent polarity has little influence on
the absorption maxima of the conjugated nucleosides. In contrast, both
emission wavelength and intensity are markedly affected by solvent
polarity. In ether, the least polar solvent tested, nucleoside 2a, for
example, displays a relatively weak emission with a maximum at 395 nm. In
water, the most polar solvent examined, 2a exhibits an intense emission
band which peaks around 430 nm and decays deeply into the visible
(>550 nm). Solvents of intermediate polarity display an intermediate
behavior with a clear emission bathochromic and hyperchromic effects with
increasing solvent polarity. Nucleoside 2a, containing a conjugated
furan, exhibits the highest sensitivity to solvent polarity (Table 2) and
is therefore selected for incorporation into oligonucleotides.

[0117]The absorption and emission spectra of the singly modified single
stranded oligonucleotide 5 are similar to those exhibited by the free
nucleoside 2a in buffer. When hybridized to its perfect complement 6, a
duplex (5.6) that is as stable as the control unmodified duplex 6.8 is
obtained (Tm=56° C. for both). Similar to other emissive
nucleosides (e.g., 2-aminopurine), the emission of the furan containing
dU is significantly quenched when found in a perfectly base paired
duplex. Importantly, thermal denaturation curves, determined by either
absorbance at 260 nm or emission at 430 nm, yield the same melting
temperature (Tm=56° C.).

[0118]Abasic sites are important DNA lesions that can be generated either
spontaneously or via enzymatic base excision of damaged nucleosides.
Several methods have been developed for detecting the presence of these
cytotoxic abasic sites, most require irreversible modifications of
isolated DNA. When oligo 5 is hybridized to the
tetrahydrofuran-containing oligo 7a duplex containing an abasic site is
formed. Remarkably, the emission of duplex 5•7 is enhanced 7-fold
when compared to the duplex 5•6, formed upon hybridization to the
perfect complement. Nucleoside 2a, when incorporated into a reporter
oligonucleotide, positively signals the presence of a DNA abasic site.

[0119]An unpaired base opposite an abasic site can be intrahelical or
extrahelical depending on the sequence context. While not being bound by
theory, the working hypothesis is that 2a is intrahelical, assuming a syn
conformation. This stacked conformation protects the hydrophobic furan
moiety, while projecting the hydrogen bonding face toward the major
groove. Support is offered by the following: (a) duplex 5-7 is more
stable than the control duplex 7•8 that contains a dT residue
opposite the abasic site (Tm=39 and 35° C., respectively). The
increased stability of the modified abasic duplex
(ΔTm=+4° C.) suggests a favorable accommodation of the
modified nucleobase by the duplex, and (b) the emission band observed for
duplex 5•7 decays sharper (>500 nm) than when compared to the
emission exhibited by the free nucleoside in solution. This is consistent
with flattening of the chromophore that can be associated with the
restricted rotation of the conjugated furan ring upon inclusion within
the DNA duplex.

[0126]All references recited are herein incorporated by reference, in
their entirety. Further, although the invention has been described with
reference to the above examples, it will be understood that modifications
and variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the following
claims.